Antiproliferative and neurotrophic molecules

ABSTRACT

Neurotrophic and antiproliferative compounds related to the antiepileptic drug valproate are provided. These compounds are useful for promoting neuronal function as in neurodegenerative disorders and for treating neoplastic disease.

FIELD OF THE INVENTION

This invention provides methods and compositions useful for theprevention and/or treatment of neurodegenerative and proliferativediseases. The compositions of the invention promote neuronal cellmaturation and retard their proliferation. In particular, this inventionrelates to nonprotein neurotrophic molecules capable of passing theblood brain barrier to provide therapeutic effects.

BACKGROUND

Proper function of the nervous system requires the maturation andmaintenance of neuronal cells. In addition, the establishment of propersynaptic connections allows for the communication between differentneurons. Defects in the survival of neurons, or the ability to maintainsynaptic connections is associated with neurodegenerative disordersincluding Alzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis (ALS), Parkinson's disease, stroke and degeneration of neuronsdue to diabetic neuropathy and trauma.

Many of the neurodegenerative disorders are associated with the loss ordegeneration of a particular class of neuronal cells. For example, inParkinson's disease dopaminergic neurons of the substantia nigradegenerate. Whereas ALS is associated with the loss of motor neurons.Wernicke-Korsakoff syndrome, commonly associated with chronicalcoholism, causes amnesia due to damage to the mammillary bodies andmedial dorsal nucleus of the thalamus. Butters N., Seminar Neurol.(1984) 4:226-244. Alzheimer's disease appears to be associated with thedegeneration of certain cholinergic neurons. The severance of axons as aresult of trauma may cause retrograde degeneration and neuronal death.

The association between neurodegeneration and the development of diseasehas prompted the search for neurotrophic agents capable of retarding,preventing, or reversing such neurodegeneration. To date, much emphasisin this area has focused on the identification and characterization ofneurotrophic polypeptides. For example, attention has been given tostudying the effects of nerve growth factor (NFG), ciliary neurotrophicfactor (CNTF), brain drive neurotrophic factor (BDNF) and others. Thegeneral neurotrophic effect of CNTF and, in particular, its trophicaction on motor neurons has led to its investigation as a useful agentin the treatment of ALS and other neurodegenerative disorders. See, forexample, Collins et al. U.S. Pat. No. 5,141,856 and Masiakowski WO91/04316 which are incorporated herein by reference. NGF which has beenshown to promote neuronal outgrowth from central cholinergic neurons hasbeen suggested as a useful agent in the treatment of Alzheimer'sdisease. Most of the neurotrophic polypeptides identified to date areactive on relatively restricted populations of neuronal cells. Whereasothers such as CNTF are active on a greater number of neuronal celltypes.

It has generally been observed that agents which induce maturation ordifferentiation of neuronal cells in culture, also inhibit theirproliferation. Normal proliferating embryonic precursors to sympatheticand sensory neurons are induced to mature and stop dividing in thepresence of certain growth factors such as NGF. The association betweenneuronal maturation or differentiation and anti-mitotic action has alsobeen observed for certain neoplastic cells which are responsive toneurotrophic factors. For example, rat pheochromocytoma, PC12, cells inthe presence of NGF develop long neurites and stop dividing. Green LAand Tischler AS, Proc. Natl. Acad. Sci. USA (1976) 72:2424-2428. Similareffects have been observed with other neuronal cells.

Cells in the nervous system give rise to a variety of potentially fatalneoplastic diseases. For example, neuroblastoma and pheochromocytoma arebelieved to arise from cells having an origin in the neural crest.Non-neuronal cells of the nervous system including glial cells,astrocytes and Schwann cells also give rise to different types oftumors. Most present agents used for chemotherapy involving neuronalcells are cytotoxic and have relatively poor specificity andpenetrability. Treatment of neoplastic disease through agents causingmaturation has been a long sought for goal. Aaronson, S. A. Science(1991) 254:1146-1153.

Although neurotrophic polypeptides may eventually prove useful fortreating certain neurodegenerative, and proliferative disorders, theyare characterized by poor bio-availability resulting from theirrelatively large size making them resistant to passing through the bloodbrain barrier. This poor penetration into the relevant target tissueraises substantial difficulties in their use for treatingneurodegenerative disorders and neoplastic disease of the centralnervous system.

The anticonvulsant sodium valproate (VPA) is a branched chain carboxylicacid effective in the treatment of primary generalized seizures,especially those of the absence type. Pinder, R. M. et al., Drugs (1977)13:81-12. Recently, VPA has been reported to be a teratogen and has beensuggested as potentially causing neural tube defects in 1% to 2% ofexposed fetuses (Robert E. and Rosa F. W., “Maternal valproic acid andneural tube defects,” Lancet (1982) 2:937). In addition, a number ofother defects are also induced by valproic acid treatment duringpregnancy (Nau et al. J. Pharmacol. Exp. Ther. (1981) 219:768-777. Spinabifida aperta, a most serious birth defect, can now also be induced byvalproic acid in an animal model (Ehlers et al., 1992 a,b). Like theneurotrophic polypeptides, valproic acid also shows very limitedtransfer into the central nervous system of the human (Löscher et al.,Epilepsia (1988) 29:311-316). For reviews of clinical and experimentalvalproic acid teratogenesis. cf. Nau et al., Pharmacol. Toxicol. (1991)69:310-321; Nau, CIBA Foundation Symposium 181, pp. 615-664; MarcelDekker, 1993.

Studies in vitro have demonstrated valproate to potently inhibit therate of neural derived cell proliferation at concentrations within itstherapeutic plasma level (Regan, C., Brain Res. (1985) 347:394-398).This antiproliferative action of valproate is restricted to a definedpoint in the G₁ phase of the cell cycle. Martin M. and Regan C., BrainRes. (1991) 554:223-228. In the presence of valproate, cells assume adifferentiated phenotype as judged by morphology, increasedcell-substratum adhesivity and decreased affinity for concanavalin Alectin coated surfaces (Martin et al., Toxic in Vitro (1988) 2:43-48;Martin et al., Brain Res. (1988) 459:131-137; Maguire and Regan, Int. J.Devl. Neurosci. (1991) 9:581-586; Regan, C., Brain Res. (1985)347:394-398. These actions of valproate are likely to be restricted tocells of the developing neural tube as, in in vivo experimental models,valproate has been shown to increase the incidence of neural tubedefects and sequester specifically into the neuroephithelium where itgenerates cellular disarray (Dencker et al., Teratology (1990)41:699-706; Ehlers et al., Teratology (1992) 45:145-151; Ehlers et al.,Teratology (1992) 46:117-130; Kao et al., Teratogen. Mutagen,Carcinogen. (1981) 1:367-382; Turner et al., Teratology (1990)41:421-442.

Hyperthermia, which induces neural tube defects (Chernoff and Golden,Teratology (1988) 37:37-42; Edwards, Teratogen. Mutagen. Carcinogen.(1986) 6:563-582; Shiota, Am J. Med. Genet. (1982) 12:281-288; Finnellet al., Teratology (1986) 33:247-252), also arrests neural cells in theG₁ phase of the cell cycle both in vivo and in vitro (martin et al.Brain Res. (1991) 554:223-228; Walsh and Morris, Teratology (1989)40:583-592); and produces similar pro-differentiative effects to thoseobserved with valproate (Martin and Regan, Brain Res. (1988)459:131-137). Thus, a coincident anti-proliferative andpro-differentiative action may identify agents which are capable ofinducing neural tube defects yet provide a basis for the development ofcompounds useful for treatment or prevention of neurodegenerativediseases.

The studies of the structure activity relationship of teratogenicvalproate-related compounds suggest a strict structural requirement forhigh teratogenic potency. Nau, H. et al., Pharmacol. & Toxicol. (1991)69:310-321. Studies of structure-activity relationships were possible asa result of previous work demonstrating that the parent drug molecule—atleast in the case of valproic acid—and not metabolite(s) provedresponsible for the teratogenic action (Nau, Fundam Appl Toxicol, (1986)6:662-668. Molecules which are highly teratogenic were reported torequire an alpha-hydrogen atom, a free carboxyl function, and branchingon carbon atom 2 with two chains containing three carbons each formaximum teratogenic activity. (Nau and Löscher, 1986, Nau and Scott,1986). Substances which do not conform with these strict structuralrequirements are of very low or negligible teratogenic activity, butstill often exhibit good anticonvulsant activity in several experimentalmodels. These compounds may therefore be valuable anti-epileptic agents(Nau et al., Neurology (1984) 34:400-402; Löscher and Nau,Neuropharmacol (1985) 24:427-435; Wegner and Nau, Neurology (1992) 42(Supp. 5):17-24; Elmazar et al., J. Pharm. Sci. (1993) 82:1255-1258.Teratogenic activity also demonstrated stereoisomeric preferencessuggesting a stereoselective interaction between the drugs and aspecific structure within the embryo.

In the case of 4-en-VPA (2-n-propyl-4-pentenoic acid) (Hauck and Nau,Toxicol Lett (1989) 49:41-48) and 4-yn-VPA (2-n-propyl-4-pentynoic acid)(Hauck and Nau, Pharm. Res. (1992) 9:850-855) the S-enantiomers provedto be more potent teratogens than the corresponding R-enantiomers. Thisstereoselective teratogenicity was due to differing intrinsicteratogenic potencies of the enantiomers, and not due to differences inpharmacokinetics as both enantiomers of a given pair reached the targettissue to the same degree, but one was more potent than the other (Haucket al., Toxicol. Lett (1992) 60:145-153). Other examples supported thepronounced stereoselectivity of the teratogenic, but not theanticonvulsant and sedative effect (Hauck et al., Life Sci. (1990)46:513-518; Nau et al., Pharmacol. & Toxicol. (1991) 69:310-321. Carbonchains connected to carbon atom 2 of valproate which were shorter orlonger than 3 carbons reduced teratogenic activity. Nau et al. Id.Valproate's antimitotic activity has been suggested as being related toits teratogenic potential rather than as a potential therapeutic asset,as the non-teratogenic valpromide analogue is not antiproliferative(Regan et al., Toxic in Vitro (1991) 5:77-82). Teratogenic analogs ofvalproate have been synthesized to date for the purpose of producingmore desirable antiepileptic agents having fewer or no side effects andhave not been suggested as being useful in their own right for othertherapeutic purposes.

Despite continued efforts to identify compounds useful for treatingneurodegenerative and proliferative disorders there is still a greatneed for useful compounds of increased efficacy and potency.

SUMMARY OF THE INVENTION

This invention provides compounds, pharmaceutical compositions andmethods useful for promoting neuronal function and inhibiting cellmitosis. Accordingly, this invention also provides methods of preventingand treating neurodegenerative and proliferative disorders.

The compounds of this invention have the general formula (I)

wherein

R¹ is —C≡CH, —CH═CH₂ or —CH₂—CH₃,

R² is a saturated, unsaturated, branched or unbranched C₁-C₃₀ alkylgroup which is optionally substituted with a C₃-C₉ aliphatic or aromaticcyclohydrocarbon or heterocyclic group.

M is a hydrogen or a metal atom. Formula I is not 2-n-propyl-4-pentynoicacid (4-yn-VPA) or 2-n-propyl-4-pentenoic acid (4-en-VPA) and when R₁ is—CH₂—CH₃, R² is C₅ to C₃₀.

This invention also provides a method of making the compounds of theinvention.

This invention also provides pharmaceutical compositions useful forinhibiting cell mitosis and/or promoting neuronal function comprisingeffective amounts of the compounds suitable for use in the treatments ofthe invention with a pharmaceutical carrier suitable for administrationto an individual

In addition, this invention relates to methods of promoting neuronalfunction and/or survival, and in particular to methods of treatingindividuals with neurodegenerative disorders. The compounds useful fortreating neurodegenerative disorders include those of formula I asdescribed above including 2-n-propyl-4-pentenoic acid and2-n-propyl-4-pentynoic acid, as well as those of formula II

wherein R³ and R⁴ are independently of one another C₁-C₃₀ saturated orunsaturated, branched and/or unbranched aliphatic hydrocarbon,optionally substituted by a C₃₋₉ aliphatic or aromatic cyclohydrocarbon,or heterocyclic group. M is hydrogen or a metal atom.

The compounds and compositions of this invention which are neurotrophicmay be used to promote the survival and function of neurons which wouldotherwise have diminished function, degenerate or die. Accordingly, inaddition to treating individuals diagnosed with a neurodegenerativedisorder, the compounds and compositions of this invention may also beused prophylactically to prevent or retard the onset ofneurodegenerative disorders in individuals identified as being at riskfor developing such disorders.

In another embodiment of this invention, the compounds and compositionsuseful for treating neurodegenerative disorders may also be used totreat proliferative disorders. The antiproliferative activity of thecompounds and compositions may be used to prevent or retard theformation of a wide variety of tumors by administering the compounds andcompositions to a person in need of treatment. This treatment isespecially useful for treating tumors of neuronal or glial origin giventhat these compounds penetrate the CNS.

It is an object of this invention to provide neurotrophic compoundsuseful for enhancing the survival of neurons and glial cells.

It is another object of this invention to provide compound andcompositions useful for promoting the expression of characteristicsassociated with mature functioning neuronal or glial cells.

By promoting the survival and function of neuronal or glial cells, it isan object to this invention to provide compounds and compositions usefulfor the prevention and/or treatment of a variety of neurodegenerativedisorders.

Another object of this invention is to provide compounds andcompositions useful for inhibiting the pathologic proliferation ofneuronal, glial or related cells.

BRIEF DESCRIPTION OF THE FIGURES DRAWINGS

FIG. 1: Dose-Response relationship of antiproliferative effect of2-n-butyl-4-pentynoic acid; and 2-n-pentyl-4-pentynoic acid. Neuro-2aneuroblastoma cells were cultured in 25 cm² flasks for 48 hours in thepresence of test medium. After 48 hours, cells were observed,photographed and harvested with trypsin for counting using ahaemocytometer. Cell number is expressed as percentage mean±SEM (n=3) ofcontrol values.

FIG. 2: Induction of neurite outgrowth of neuro-2a neuroblastoma cells.Neuroblastoma cells were cultured in the presence of2-n-butyl-4-pentynoic acid (1.0 mM, 2mM); and 2-n-pentyl-4-pentynoicacid (0.3 mM, 0.5 mM). Test medium was added to cells after 24 hours inculture and maintained as a test medium for 48 hours after which theywere fixed in 2.5% glutaraldehyde and 0.5M sodium phosphate bufferovernight at 4° C. Cells were postfixed with osmium tetroxide andprepared for scanning microscopy as described. Fixed and stained cellswere observed in a scanning electron microscope at an acceleratingvoltage of 15 kV.

FIGS. 2A-2E: Induction of neurite outgrowth of neuro- 2a neuroblastomacells. Neuroblastoma cells were cultured in the presence of 2 -n-butyl-4 -pentynoic acid ( 1.0 mM, 2 mM); and 2 -n-pentyl-pentynoic acid ( 0.3mM, 0.5 mM). Test medium was added to cells after 24 hours in cultureand maintained as a test medium for 48 hours after which they were fixedin 2.5 % glutaraldehyde and 0.5 M sodium phosphate buffer overnight at4° C. Cells were postfixed with osmium tetroxide and prepared forscanning microscopy as described. Fixed and stained cells were observedin a scanning electron microscope at an accelerating voltage of 15 kV.

FIG. 3: Neural Cell Adhesion Molecule (NCAM) immunofluorescence inneuro-2a neuroblastoma cells. Panel A. Cells gown in the presences of2-n-pentyl-pentynoic acid (1.0 mm) show increased immunofluorescencedirected against NCAM compared to control cells. Panel B. Neuroblastomacells were cultured for 48 hours in the presence of increasingconcentrations of 2-n-butyl-4-pentynoic acid and 2-n-pentyl-4-pentynoicacid. They were then fixed and prepared for staining with rabbitanti-NCAM antibody. A second anti-rabbit antibody conjugated torhodamine was incubated with the cells to detect bound anti-NCAMantibody. Cells were observed with a fluorescence microscope at anexcitation wavelength of 535 nm. Immunofluorescence is expressed asmean±SEM.

FIGS. 3A- 1, 3A- 2 and 3B: Neural Cell Adhesion Molecule (NCAM)immunofluorescence in neuro- 2a neuroblastoma cells. Panel A. Cellsgrown in the presences of 2 -n-pentynoic acid ( 1.0 mm) show increasedimmunofluorescence directed against NCAM compared to control cells.Panel B. Neuroblastoma cells were cultured for 48 hours in the presenceof increasing concentrations of 2 -n-butyl- 4 -pentynoic acid and 2-n-pentyl- 4 -pentynoic acid. They were then fixed and prepared forstaining with rabbit anti-NCAM antibody. A second anti-rabbit antibodyconjugated to rhodamine was incubated with the cells to detect boundanti-NCAM antibody. Cells were observed with a fluorescence microscopeat an excitation wavelength of 535 nm. Immunofluorescence is expressedas mean +/− SEM.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to derivatives of valproic acid, methods of theirpreparation and pharmaceutical compositions comprising these compounds.This invention also relates to a method of promoting neuronal functionand differentiation which is useful for preventing and treatingneurodegenerative disorders. The anti-mitotic activity of the compoundsand compositions of the invention are useful for arresting cells in aspecific stage of the cell cycle and for the prevention and treatment ofneoplastic disease.

The objects of this invention are accomplished by providing potentteratogenic analogs of valproic acid which penetrate the CNS asneurotrophic/neuroprotective agents capable of treating and retardingthe onset of neurodegenerative diseases. The compounds and compositionsof this invention are also useful for controlling the cell proliferativerate and the metastatic potential of neoplastic or potentiallyneoplastic cells.

Accordingly, the compounds of this invention have the general formula(I)

wherein

R¹ is —C≡CH, —CH═CH₂, or —CH₂—CH₃,

R₂ is independently a saturated, unsaturated with at least one double ortriple bond, branched or unbranched C₁₋₃₀ alkyl group, optionallysubstituted with an aliphatic or aromatic C₃₋₉ cyclohydrocarbon orheterocyclic group; with the proviso that when R¹ is CH₂—CH₃, R² isC₅₋₃₀, and that formula I is not 2-n-propyl-4-pentynoic acid or2-n-propyl-4-pentenoic acid (4-en-VPA).

M is a hydrogen or a metal atom.

This invention also includes the racemic mixtures and the separateenantiomeric R and S forms of the compounds and pharmaceuticalacceptable salts thereof.

Preferably, R¹ is —C≡CH and R² is an unbranched saturated C₂-C₁₀ alkylgroup. More preferred, R² is an unbranched, saturated C₄-C₆alkyl group.Examples of preferred substituents for R²include —(CH₂)₁₋₉—CH₃, morepreferred is —(CH₂)₃₋₆—CH₃, and most preferred is —(CH₂)₄₋₅—CH₃. Mostpreferred compounds are 2-n-butyl-4-pentynoic acid (R¹=—C≡H;R²=—(CH₂)₃—CH₃)), 2-n-pentyl-4-pentynoic acid (R¹=—C≡H; R²=—(CH₂)₄—CH₃)and 2-n-hexyl-4-pentynoic acid (R¹=—C≡H; R²=—(CH₂)₅—CH₃). In addition,although both enantiomers and their racemic mixtures are consideredwithin the scope of this invention, the S-enantiomeric form ispreferred. Preferred metal atoms are sodium or other alkali metals, aswell as alkaline earth metals such as, for example, calcium ormagnesium.

The teratogenic, antiproliferative and prodifferentiative potencies ofthe preferred compounds are much higher than of the antiepileptic drugvalproic acid.

Further branching of R¹ or R² reduces the potency of the correspondingcompounds. This is demonstrated by the low teratogenic,antiproliferative and prodifferentiative potency of the followingcompound.

Unsaturation between C₂ and C₃ (IV) as well as methylation of the C₅ (V,VI) also lowers, but does not abolish, the above mentioned cellularneurotrophic and antiproliferative activity

In agreement with our basic hypothesis, compound IV (Nau et al.,Neurology (1984) 34:400-402; Nau and Löscher, Fundam Appl. Toxicol.(1986) 6:669-676; Nau and Scott, Nature (1986) 323:276-278; Vorhees etal., Teratology (1991) 43:583-590; Ehlers et al., Devel. Pharmacol.Ther. (1992) 19:196-204 and VI (Nau et al., Pharmacol. & Toxicol. (1991)69:310-321; Elmazar et al., J. Pharm. Sci. (1993) 82:1255-1258 has verylow or undetectable teratogenic activities, but good anticonvulsantproperties in experimental models.

The compounds and compositions of this invention are more potentteratogenic analogues of valproate and exhibit greater antiproliferativeand neurotrophic/neuroprotective activity than the parent. In contrastto saturated valproate analogues (where both chains must contain 3carbon atoms each for maximal activity) a double or triple bond in the ωposition of one chain exhibits higher activities when the other chaincontains 4 to 6 carbon atoms. The 2-n-propyl-4-pentynoic acid,2-n-butyl-4-pentynoic acid, 2-n-pentyl-4-pentynoic acid and2-n-hexyl-4-pentynoic acid are the most potent valproate-relatedteratogens synthesized. 2-n-butyl-4-pentynoic acid,2-n-pentyl-4-pentynoic and 2-n-hexyl-4-pentynoic acid are morepreferred. Most preferred are 2-n-pentyl-4-pentynoic acid and2-n-hexyl-4-pentynoic acid.

The preferred compounds for use with this invention possess a chiralalpha-carbon. As a result of chirality, the efficacy and potency ofdifferent enantiomeric forms may differ. For example,S-2-n-propyl-4-pentynoic acid has significantly greater teratogenicpotential than the R-enantiomeric form. Hauck and Nau, Pharm. Res.(1992) 9:850-855; Hauck et al. Toxicol. Lett. (1992) 60:145-153. See Nauet al. Pharmacol. Toxicology (1991) 69:310-321 which is incorporatedherein by reference. Although there is no general rule of theabove-identified compounds, the S enantiomeric form is preferred.

The compounds of this invention are prepared by reacting anappropriately substituted malonic acid diethylester with an appropriateunsaturated alkylating agent such as a straight-chain alkylhalide. Theproduct is then hydrolyzed and decarboxylated.

The reaction can also be carried out in the reciprocal manner in that amalonic acid diethylester, substituted with an unsaturated function isreacted with an appropriate alkylhalide. This reaction is again followedby hydrolysis and decarboxylation.

The novel compounds of this invention may be produced according to themethod of this invention. In one embodiment, the method of synthesizingthe compounds comprises combining a malonic acid diester reactant with afirst halide reactant having the general formula

R²—X   (VII)

wherein R² is a saturated or unsaturated branched or unbranched C₁-C₃₀alkyl group and X is a halide. This first reaction produces a2-alkyl-malonic acid diester. The 2-alkyl-malonic acid diester is thenfurther combined with a second halide reactant having the generalformula

R¹—CH₂—X   (VIII)

wherein R¹ is —C≡CH, —CH═CH₂or —CH₂—CH₃ to produce compounds with thegeneral formula

wherein R⁵ is an alkyl group.

The resulting diesters are then hydrolyzed, decarboxylated andoptionally converted into a salt.

In an alternative embodiment, the order of carrying out the reactions isreversed, such that the R¹—CH₂—X is combined with the malonic aciddiester followed by further reaction with the R²—X.

In a preferred method of preparing the compounds of this invention,malonic acid diethylester is treated with a base, for example, sodiumethylate, to deprotonate carbon 2. Subsequent treatment of the resultingdeprotonated ester with an alkylating agent in the form of astraight-chain alkyl halide yields a 2-n-alkyl-malonic aciddiethylester.

This product is further alkylated with sodium ethylate and either2-propynehalide to yield XI

 or 2-propenehalide to yield XII

 or 2-propylhalide to yield XIII

The diesters (XI) and (XII) and (XIII) are hydrolyzed and decarboxylatedwith potassium hydroxide in ethanol/water with heat treatment.

Another embodiment of this invention is the promotion of neural functionby contacting neural cells with a neurotrophic amount of a compound offormula (II)

wherein R³ and R⁴ are independently of each other saturated orunsaturated, branched, or unbranched, C₁-C₃₀ aliphatic hydrocarbons,optionally possessing at least one double or triple bond. Preferably R³and R⁴ are unbranched, and R³ is less than or equal to a three carbonchain. R⁴ preferably is a saturated alkyl group and is preferably fromC₂-C₁₀, as in for example —(CH₂)₁₋₉—CH₃, and more preferably from C₄ toC₆, as in for example —(CH₂)₃₋₅—CH₃. In addition to the compounds statedabove in connection with formula I, other compounds which are useful forthe promotion of neuronal function and inhibition of cell mitosis aredescribed in Nau et al. PCT application PCT/DE93/00861 publish asWO94/06743, and which is incorporated herein by reference.

Preferred compounds useful for promoting neuronal function include forexample, 2-n-propyl-4-pentynoic acid (R³=—CH₂—C≡CH; R⁴=—(CH₂)₂—CH₃);valproic acid (R³=R⁴=—(CH₂)₂—CH₃); 2-n-propylhexanoic acid(R³=—(CH₂)₃—CH₃, R⁴=—(CH₂)₂—CH₃); and 2-n-butylhexanoic acid(R³=—(CH₂)₃—CH₃).

The promotion of neuronal function is particularly useful for preventingand treating neurodegenerative disorders. Neurodegenerative disordersinclude any disorder resulting in neuronal degeneration which isresponsive to at least one of the valproate analogues or valproateitself.

The neurotrophic activity associated with valproate and its analoguesmay be determined based on in vitro indices of differentiation,including inhibition of mitosis, increase in neurite outgrowth, and NCAMexpression. For example, the ability to promote neurite outgrowth iscorrelated with enhanced survival of certain cultured neural cellsincluding embryonic sensory and sympathetic neurons. Proliferatingimmature neuroblasts, in vitro, have a rounded shape and are looselyadherent to culture surfaces. In the presence of a neurotrophic factor,these cells become more adherent and sprout processes known in the artas neurites. Accordingly, in vitro neurite outgrowth may be used as anassay for determining concentrations of compound in contact with targetcells which would be expected to achieve desirable neuroprotectingeffects.

Methods of assessing neurite outgrowth in vitro are well known in theart and, for example, may be assessed through direct microscopic visualinspection or through the use of computer aided image processing.

Another characteristic of neurotrophic factors which may be used toassess the neuroprotective action of the compounds and compositions ofthis invention is their ability to promote survival or certain specificcell types. For example, NGF is required in vitro for the survival ofcertain specific cell types which die in the absence of NGF. Such NGFdependent cells include neurons of the chick dorsal rat ganglia at aboutembryonic day E5to E8.

Scanning electron microscopy illustrates the cells ability to increasecell-substratum adhesivity. They eliminate rounded and clustered growth,typical of tumor cells, and induce a flattening and greater interactionwith the substratum (FIG. 2). In vivo, it is generally believed thatthese neurites further differentiate into axons and dendrites and formsynapses with other neurons. During diseases involvingneurodegeneration, there may be a loss of synapses and degeneration ofaxons and dendrites resulting in a deficit of neuronal function.

Another index of differentiation resulting from the neurotrophicactivity of valproate analogues is an increase in NCAM expression.Further, increases in NCAM prevalence enhances neurite outgrowth.Doherty et al., Nature (1990) 343:464-466. NCAM has been reported asplaying a fundamental role in memory formation as intraventricularinfusion of anti-NCAM during consolidation of a recent learning eventinduces an amnesia. Doyle et al., J. Neurochem. (1992) 59:1570-1573,which is incorporated herein by reference. Rapid endocytosis of theAplysia NCAM homologue was reported following a serotonin-induced changein synapse structure in vitro. Bailey et al., Science (1992)256:645-649.

During development of individual brain regions, or in adults exhibitingongoing neurogenesis, NCAM transiently increases its sialylation state.See review, Regan, Int. J. Biochem. (1991) 23:513-523, which isincorporated herein by reference, Rougon (1993) Eur. J. Cell Biol.61:197-207. The synapse specific NCAM isoform (NCAM 180) which isassociated with differentiated neurons increases its sialylation stateduring later stages of development until the period of synaptogenesis iscomplete. Breen et al., J. Neurochem (1988) 50:712-716. A similarisoform-specific sialylation of NCAM 180 occurs during consolidation ofa passive-avoidance response. Doyle et al., J. Neurosci Res., (1992)31:513-523.

Accordingly, the methods of treatment and prevention ofneurodegenerative diseases rely on the ability of valproate and itsanalogues to possess neurotrophic activity such as promoting neuriteoutgrowth and survival of neuronal cells and NCAM expression.

It is contemplated that the methods of treatment may provide benefits topersons with neurodegeneration from disorders including, but not limitedto ALS, Alzheimers disease, Parkinson's disease, Huntington's disease,diabetic neuropathy and stroke. In addition, the neurite promotingactivity of the disclosed compounds and compositions would also providebenefits to individuals with traumatic nerve injury.

IN another embodiment of this invention, methods are provided forarresting cells in a specific stage of the cell cycle which leaves thecells in a differentiated state by contacting cells with a mitoticinhibitory amount of a compound of formula II as described above.Preferred substituents for R³, R⁴ and M for inhibiting mitosis are thesame as those for promoting neuronal function, with the proviso thatformula II is not valproate if simply used to inhibit cell mitosis.Preventing mitosis in this manner is useful for enhancing the expressionof specific proteins associated with the differentiated phenotype. Thisenhanced expression facilitates purification of such proteins. Inaddition, arresting or retarding mitosis is useful for treatingproliferative disorders by administering to individuals in need oftreatment valproate and/or another of its anti-mitotic analogues.

We have observed sensitivity to valproate or its antimitotic analoguesin all cells tested. Such cell types include: primary astrocytes, humanastrocytoma, and those from cardiac, renal, and immune systems.Accordingly, the antiproliferative action of valproate and its otheranalogues described herein should have broad applicability for a widevariety of tumors derived from a variety of cell types and particularlythose mentioned above.

The neurotrophic and/or anti-mitotic effective amounts of valproate andactive analogues may be determined using standard dose-response curves.Accordingly, representative cells may be cultured in vitro in thepresence of varying concentration of test compound. At an appropriatetime, the cells under the different conditions are examined for theappropriate parameter (for example, cell number for antimitoticactivity; neurite outgrowth for neurotrophic activity) and the ED₅₀ maybe determined.

The preferred compounds of this invention exert a most profoundantiproliferative action with ED50 values well below (<0.5 mM) thoseobserved with valproate (FIG. 1). Thus, these compounds may be expectedto act an concentrations which will be devoid of the sedative andhepatotoxic side effects of valproate. The preferred compounds alsoexert the prodifferentiative action observed with valproate. In theneuro-2a neuroblastoma cell line they induce a marked neuritogenicresponse which correlates with their antiproliferative potential (FIG.2).

In addition, the more potent of these compounds increase neural celladhesion molecule (NCAM) prevalence (FIG. 3). This cell recognitionsystem regulates neural plasticity during development and, later, duringinformation storage in the adult animal by altering its prevalence andglycosylation state (Doyle et al., J. Neurosci Res., (1992) 31:513-523).Drugs which reverse scopolamine-induced amnesia, such aspiracetam-related compounds, appear to act through a neuroprotectivemechanism which involves a non-specific increase in NCAM glycosylationand/or prevalence (Doyle et al., J. Neurochem. (1993) 61:266-272).Consequently agents which would induce NCAM expression may be predictedto have neuroprotective potential.

This invention also provides pharmaceutical compositions useful fortreating neurodegenerative or proliferative disorders comprising acompound selected from formulas I or II as described above. In additionto the compounds of formula I or II, the pharmaceutical composition mayalso comprise adjuvant substances and carriers. The compositions may bein the form of tablets, capsules, powders, granules, lozenges,suppositories, reconstitutable powders, or liquid preparations such asoral or sterile parenteral solutions or suspensions.

In order to obtain consistency or administration it is preferred that acomposition of the invention is in the form of a unit dose.

Unit dose presentation forms for oral administration may be tablets andcapsules and may contain conventional excipients such as binding agents,for example syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone, fillers, for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; disintegrants, for examplestarch, polyvinylpyrrolidone, sodium starch glycolate ormicrocrystalline cellulose; or pharmaceutically acceptable wettingagents such as sodium lauryl sulphate.

The solid oral compositions may be prepared by conventional methods ofblending, filling, tabletting or the like. Repeated blending operationmay be used to distribute the active agent throughout those compositionsemploying large quantities of fillers. Such operations are of courseconventional in the art. The tablets may be coated according to methodswell known in normal pharmaceutical practice, in particular with anenteric coating.

Oral liquid preparations may be in the form of, for example, emulsions,syrups, or elixirs, or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, for example sorbitol syrup, methyl cellulose,gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminumstearate gel, hydrogenated edible fats; emulsifying agents, for examplelecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (whichmay include edible oils), for example almond oil fractionated coconutoil, oily esters such as esters of glycerine, propylene glycol, or ethylalcohol; preservatives, for example methyl or propyl p-hydroxybenzoateof sorbic acid; and if desired conventional flavoring or coloringagents.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, and, depending on theconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the compound can be dissolved in water forinjection and filter sterilized before filling into a suitable vial orampoule and sealing. Advantageously, adjuvants such as a localanaesthetic, a preservative and buffering agents can be dissolved in thevehicle. To enhance the stability, the composition can be frozen afterfilling into the vial and the water removed under vacuum. Parenteralsuspensions are prepared is substantially the same manner, except thatthe compound is suspended in the vehicle instead or being dissolved, andsterilization cannot be accomplished by filtration. The compound can besterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of thecompound.

The dose of the compound used in the treatment of such disease will varyin the usual way with the seriousness of the disorders, the weight ofthe sufferer, and the relative efficacy of the compound.

Antiproliferative and neuroprotective actions should be sufficient toachieve the desired inhibition of mitosis or neuroprotection withoutserious hetaptotoxic side effects. The plasma concentrations to beachieved will be sufficient to provide therapeutically effectiveconcentrations of compound in contact with the target cells. Standardclinical techniques may be used to determine the effective amount ofcompound to be administered to achieve the desired therapeutic effect.Dose response curves may be determined first in vitro in a relevantanimal model to determine ranges of expected therapeutic concentrationsin humans. For example, mitosis of mouse neuro-2a-neuroblastoma cells isinhibited by valproate with an ED₅₀ of 1.0-1.3 mM. Other cell lines,including those of human origin may be used to assesses activity aswell.

EXAMPLE 1

0.1 mol n-butyl malonic acid diethylester and 0.1 mol 3-bromo-1-propinewere placed in a dry argon flushed flask and heated to 60° C. To thismixture was added 0.1 mol sodium ethanolat (prepared from 0.1 mol sodiumand 50 mol dry ethanol) dropwise such as to keep the mixture boiling.After completion of the addition, the mixture was heated until TLC(Silica alu sheets, hexane/ethylacetate 7.5/1) showed absence ofstarting material (usually 1-2 hours). The ethanol was evaporated underreduced pressure, the remaining salts were dissolved in water and theproduct was extracted three times with CH₂Cl₂. The organic phase wasdried over sodium sulfate and evaporated The distillation under reducedpressure resulted in the unsymmetrically substituted malonic aciddiethylester.

bP_(0.3) mbar:78°-82° C.

The dialkylated malonic esters were heated in a solution of 20.3 g (0.35mol) potassium hydroxide, 50 ml water and 100 ml ethanol. Aftercompletion of the saponification, ethanol was evaporated under reducedpressure. The remaining residue was diluted with water and washed withether. The water layer was acidified with concentrated HCl (pH<2) andextracted with ether. Drying over anhydrous sodium sulfate andconcentration under reduced pressure yielded crude dialkyl malonic acid.Decarboxylation was achieved by heating of the crude product (120°-180°C.). The dark residue was distilled twice in vacuo resulted in thedesired products.

Overall yield: 18%

bP_(0.1) mbar: 75°-78° C.

1_(H-NMR) (CDCl₃): 0.94 (3H, t, CH₃), 1.34 (4H, m, 2×CH₂), 1.72 (2H, m,CH₂—CHRCOOH), 2.04 (1H, t, C≡C—H), 2.36-2.68 (3H, m, CHRCOOH—CH₂—C≡C),11.88 (1H, s, broad, COOH)

EXAMPLE 2

0.1 mol n-pentyl malonic acid diethylester is reacted with 0.1 mol3-bromo-1-propine as described in example 1.

Overall yield: 14%

bP₁₅ mbar: 135° C.

1_(H-NMR) (CDCL₃): 0.92 (3H, t, CH₃), 1.32 (6H, m, 3=CH₂), 1.72 (2H, m,CH₂—CHRCOOH), 2.04 (1H, t, C≡C—H), 2.40-2.72 (3H, m, CHRCOOH—CH₂—C≡C),11.32 (1H, s, broad, COOH)

EXAMPLE 3

(±)-2-(2-propinyl)-Octanoic acid (Hexyl-4-yn)

Synthesis is by the dianion method (Petragnani, Synthesis 521, 1982).

All glassware was oven dried and the reaction apparatus was flushed withargon throughout the entire operation.

Lithium-dianion (0.2 Mol) was prepared by adding 0.2 Mol n-butyl-lithiumto a solution of 0.2 Mol freshly distilled diisopropylamine and 130 mldry tetrahydrofurane at 0° C. Octanoic acid (0.1 Mol) was added followedby 19 hexamethylphosphoric acid triamide to effect solution of thedianion. The resulting mixture was stirred at room temperature for 30min followed by cooling to −60° C. and addition of 3-bromo-1-propin (0.1Mol) quickly via a syringe. The temperature rose instantly. Aftercooling back to −60° C., the reaction was stirred and monitored by TLC(Hexane:Ethylacetate=7.5:1 plus 5% acetic acid) until completion (ca 1.5h). Cooling was removed and 200 ml 10% HCl was added. The phases wereseparated and the water phase was extracted twice with ether. Thecombined organic phases were washed with half saturated NaCl solutionand dried with Na₂SO₄. Evaporation of the solvent yielded a yellow oil.Destillation yielded a colorless liquid (bp. 82°-84° C., 0.1 mbar). ¹HNMR (CDCl₃)=0.88 (3H, t, CH₃), 1.40 (8H, mc, CH₂), 1.90 (2H, mc, CH₂),2.04 (1H, t, ≡—H), 2.32-2.68 (3H, m, CH₂, H_(α)), 12.04 (1H, s broad,COOH).

EXAMPLE 4

The following non-limiting preferred examples are compounds within thescope of this invention:

2-n-propyl-4-pentynoic acid

2-n-prop-1¹-enyl-4-pentynoic acid

2-n-prop-2¹-enyl-4-pentynoic acid

2-i-propyl-4-pentynoic acid

2-i-propenyl-4-pentynoic acid

2-n-butyl-4-pentynoic acid

2-n-but-1¹-enyl-4-pentynoic acid

2-n-but-2¹-enyl-4-pentynoic acid

2-n-but-3¹-enyl-4-pentynoic acid

2-(1¹-methylbutyl)-4-pentynoic acid

2-(1¹-methylprop-1¹-enyl)-4-pentynoic acid

2-(1¹-methylprop-2¹-enyl)-4-pentynoic acid

2-(2¹-methylpropyl)-4-pentynoic acid

2-(2¹-methylprop-1¹-enyl)-4-pentynoic acid

2-(2¹-methylprop-2¹-enyl)-4-pentynoic acid

2-tert.-butyl-4-pentynoic acid

2-n-pentyl-4-pentynoic acid

2-(1¹-methylbutyl)-4-pentynoic acid

2-(2¹-methylbutyl)-4-pentynoic acid

2-(3¹-methylbutyl)-4-pentynoic acid

2-(1¹,1¹-dimethylpropyl)-4-pentynoic acid

2-(1¹,2¹-dimethylpropyl)-4-pentynoic acid

2-(2¹,2¹-dimethylpropyl)-4-pentynoic acid

2-n-hexyl-4-pentynoic acid

2-n-hex-1¹-enyl-4-pentynoic acid

2-n-hex-2¹-enyl-4-pentynoic acid

2-n-hex-3¹-enyl-4-pentynoic acid

2-n-hex-4¹-enyl-4-pentynoic acid

2-n-hex-5¹-enyl-4-pentynoic acid

2-(1¹-methylpentyl)-4-pentynoic acid

2-(1¹-methylpent-1¹-enyl)-4-pentynoic acid

2-(1¹-methylpent-2¹-enyl)-4-pentynoic acid

2-(1¹-methylpent-3¹-enyl)-4-pentynoic acid

2-(1¹-methylpent-4¹-enyl)-4-pentynoic acid

2-(2¹-methylpentyl)-4-pentynoic acid

2-(2¹-methylpent-1¹-enyl)-4-pentynoic acid

2-(2¹-methylpent-2¹-enyl)-4-pentynoic acid

2-(2¹-methylpent-3¹-enyl)-4-pentynoic acid

2-(2¹-methylpent-4¹-enyl)-4-pentynoic acid

2-(3¹-methylpentyl)-4-pentynoic acid

2-(3¹-methylpent-1¹-enyl)-4-pentynoic acid

2-(3¹-methylpent-2¹-enyl)-4-pentynoic acid

2-(3¹-methylpent-3¹-enyl)-4-pentynoic acid

2-(3¹-methylpent-4¹-enyl)-4-pentynoic acid

2-(4¹-methylpentyl)-4-pentynoic acid

2-(4¹-methylpent-1¹-enyl)-4-pentynoic acid

2-(4¹-methylpent-2¹-enyl)-4-pentynoic acid

2-(4¹-methylpent-3¹-enyl)-4-pentynoic acid

2-(4¹-methylpent-4¹-enyl)-4-pentynoic acid

2-(1¹,1¹-dimethylbutyl)-4-pentynoic acid

2-(1¹,1¹-dimethylbut-2¹-enyl)-4-pentynoic acid

2-(1¹,1¹-dimethylbut-3¹-enyl)-4-pentynoic acid

2-(1¹,2¹-dimethylbutyl)-4-pentynoic acid

2-(1¹,2¹-dimethylbut-1¹-enyl)-4-pentynoic acid

2-(1¹,2¹, dimethylbut-2¹-enyl)-4-pentynoic acid

2-(1¹,2¹, dimethylbut-3¹-enyl)-4-pentynoic acid

2-(1¹,3¹-dimethylbutyl)-4-pentynoic acid

2-(1¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid

2-(1¹,3¹-dimethylbut-2¹-enyl)-4-pentynoic acid

2-(1¹,3¹-dimethylbut-3¹-enyl)-4-pentynoic acid

2-(2¹,2¹-dimethylbutyl)-4-pentynoic acid

2-(2¹,2¹-dimethylbut-3¹-enyl)-4-pentynoic acid

2-(2¹,3¹-dimethylbutyl)-4-pentynoic acid

2-(2¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid

2-(2¹,3¹-dimethylbut-2¹-enyl)-4-pentynoic acid

2-(2¹,3¹-dimethylbut-3¹-enyl)-4-pentynoic acid

2-(3¹,3¹-dimethylbutyl)-4-pentynoic acid

2-(3¹,3¹-dimethylbut-1¹-enyl)-4--pentynoic acid

2-(1¹,1¹,2¹-trimethylpropyl)-4-pentynoic acid

2-(1¹,1¹,2¹-trimethylprop-2¹-enyl)-4-pentynoic acid

2-(1¹,2¹, 2¹-trimethylpropyl)-4-pentynoic acid

2-n-heptyl-4-pentynoic acid

2-(1¹-methylhexyl)-4-pentynoic acid

2-(2¹-methylhexyl)-4-pentynoic acid

2-(3¹-methylhexyl)-4-pentynoic acid

2-(4¹-methylhexyl)-4-pentynoic acid

2-(5¹-methylhexyl)-4-pentynoic acid

2-(1¹,1¹-dimethylpentyl)-4-pentynoic acid

2-(1¹,2¹-dimethylpentyl)-4-pentynoic acid

2-(1¹,3¹-dimethylpentyl)-4-pentynoic acid

2-(1¹,4¹-dimethylpentyl)-4-pentynoic acid

2-(2¹,2¹-dimethylpentyl)-4-pentynoic acid

2-(2¹,3¹-dimethylpentyl)-4-pentynoic acid

2-(2¹,4¹-dimethylpentyl)-4-pentynoic acid

2-(3¹,3¹-dimethylpentyl)-4-pentynoic acid

2-(3¹,4¹-dimethylpentyl)-4-pentynoic acid

2-(4¹,4¹-dimethylpentyl)-4-pentynoic acid

2-(1¹,1¹,2¹-trimethylbutyl)-4-pentynoic acid

2-(1¹,1¹,3¹-trimethylbutyl)-4-pentynoic acid

2-(1¹,2¹,3¹-trimethylbutyl)-4-pentynoic acid

2-(2¹,2¹,3¹-trimethylbutyl)-4-pentynoic acid

2-(2¹,3¹,3¹-trimethylbutyl)-4-pentynoic acid

EXAMPLE 5

Maintenance of Cell Lines.

The mouse neuro-2a neuroblastoma cell line (Klebe and Ruddle, 1969 J.Cell Biol., 43:69A) was cultured in Dulbecco's modified Eagle's medium(DMEM; Flow Laboratories) supplemented with 10% fetal bovine serum(Tissue Culture Services), 200 mM glutamine and 100 μg/ml of gentamicinor 100 units/ml and 100 μg/ml of penicillin/streptomycin antibiotics(Sigma Chemicals). The cells were maintained in a water-humidifiedatmosphere of 9% CO₂at 37° C. Cells were passaged using 0.025% trypsin(Gibco) in DMEM, and were seeded at a density of 1×10⁴ cells/cm².

Antiproliferative Assay.

Neuro-2a cells were seeded in 25cm² flasks (Costar) at a density of1×10⁴ cells/cm². Following a recovery period of 24 h, the agent to beexamined was added to the cells in a vehicle of dimethyl sulphoxide(DMSO), the volume of which was 0.2% of the total volume of mediumbathing the cells. A flask containing the DMSO vehicle alone wasemployed as control. Following incubation for 48h, cells were examinedusing an inverted phase contrast microscope (Leitz Diavert) andphotographed (Ilford 50ASA film). Cells were then harvested bytrypsinization and were counted using a haemocytometer (improvedNeubauer model). FIG. 1 shows the resultant decrease in cellproliferation.

Scanning Electron Microscopy.

Cells which were to be examined by scanning electron microscopy weregrown as previously described in 25cm² flasks. Following 48 h exposureto the agent, cells were fixed in a solution of 2.5% glutaraldehyde in0.1M sodium phosphate buffer, pH 7.4, overnight at 4° C. The cells werepost-fixed subsequently in phosphate-buffered 1% osmium tetroxide for 1h at room temperature, washed and were dehydrated gradually for 1 hourusing a series of ethanol concentrations stepwise from 20, 40, 60, 80 toa final concentration 100%.

Sections of the base of the tissue culture flask were removed and werecritical point dried to minimize shrinkage and cracking. This wasachieved by placing the samples in a Polaron critical point dryer andpurging the chamber several times with CO₂ to remove all traces ofethanol. After 1 h the temperature and pressure were increased to 40° C.and 1200 lbs/in², respectively, at which stage the critical point forcarbon dioxide had been reached and drying was completed.

Specimens were subsequently removed from the chamber, mounted on stubssuitable for scanning electron microscopy using conductive carbon cement(Neubauer) and were sputter coated with gold under vacuum (5×10⁻² torr)in the presence of argon gas at a current of 20 mA for 3 minutes(Polaron E5100). Following gold-coating, samples were examined in thescanning electron microscope (JEOL 35C) at an accelerating voltage of 15kV. Images were recorded on film (Kodak Plus-X Pan 120 film) as shown inFIG. 2.

Fluorescence Microscopy.

Cells were seeded in 24-well plates at a density of 1×10⁴ cells/cm².Following a recovery period 24 h they were exposed for an additional 48h to the drug under investigation. Cells were progressively fixed by sixten-minute incubations with DMEM containing increasing concentrations ofneutral buffered formalin stepwise from 10, 30, 50, 70, 90 to a finalconcentration of 100%. When fixation was complete, cells were washedthree times with phosphate buffered saline pH 7.4 over a 30 minuteperiod. The cells were then incubated with a 1 in 50 dilution of rabbitanti-NCAM antibody, (Pliophys et al. J. Neuropsychiatr. 2:413-417, 1990)in phosphate buffered saline containing 1% (W/V) bovine serum albuminfor 1 h at RT and washed three times with phosphate buffered saline, pH7.4 for 30 minutes. Washed cells were then incubated for 1 h at RT withthe secondary anti-rabbit antibody diluted 1 in 50 in phosphate bufferedsaline containing 1% (W/V) bovine serum albumin (Sigma) which wasconjugated to rhodamine. The cells were again washed three times withphosphate buffered saline pH 7.4 and were then mounted using Citifluor(Agar Scientific) containing a fluorescence enhancer. Fluorescence ofrhodamine was visualized using an excitatory wavelength of 535 nm (Leicafilter block N2.1) on a Leitz DMRB fluorescence microscope. Fluorescenceintensity was examined at points of cell-cell contact using a Quantimet500 Image Analysis System. Fluorescence intensity is expressed as greylevel at points of cell contact relative to that observed in thecontrol. FIG. 3 shows the increase in NCAM immunofluorescence.

While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that the basic constructions can be altered toprovide other embodiments which utilize the methods of this invention.Therefore, it will be appreciated that the scope of this invention isdefined by the claims appended hereto rather than by the specificembodiments which have been presented hereinbefore by way of example.

We claim:
 1. Compounds of formula (I)

wherein R¹ is —C≡CH, —CH═CH₂, or —CH₂—CH₃, R² is a saturated,unsaturated, branched and/or unbranched C₁-C₃₀ alkyl group, optionallyfurther comprising an aliphatic or aromatic C₃₋₉ cyclohydrocarbon orheterocyclic group comprising 3 to 9 atoms; and M is a hydrogen or ametal atom, and enantiomeres thereof and pharmaceutical acceptable saltsthereof; with the proviso that the compound of formula (I) is not2-n-propyl-4-pentynoic acid, 2-benzylpentanoic acid, 4,4′-dien-valproicacid or 2-n-propyl-4-pentenoic acid (4-en-VPA), and when R¹ is —CH₂—CH₃,R² is C₅ to C₃₀.
 2. The compounds according to claim 1 wherein R¹ is—C≡CH or —CH═CH₂ and R² is unbranched and —(CH₂)₁₋₉—CH₃.
 3. The compoundaccording to claim 2 wherein R² is —(CH₂)₃₋₆—CH₃.
 4. The compoundsaccording to claim 2 wherein R² is —(CH₂)₄₋₅—CH₃.
 5. The compoundsaccording to claim 4 wherein R¹ is —C≡CH.
 6. The S enantiomer of thecompounds according to claim
 1. 7. The compounds according to claim 1,selected from the group consisting of: 2-n-prop-1¹-enyl-4-pentynoic acid2-n-prop-2¹-enyl-4-pentynoic acid 2-i-propyl-4-pentynoic acid2-i-propenyl-4-pentynoic acid 2-n-but-1¹-enyl-4-pentynoic acid2-n-but-2¹-enyl-4-pentynoic acid 2-n-but-3¹-enyl-4-pentynoic acid2-(1¹-methylbutyl)-4-pentynoic acid2-(1¹-methylprop-1¹-enyl)-4-pentynoic acid2-(1¹-methylprop-2¹-enyl)-4-pentynoic acid2-(2¹-methylpropyl)-4-pentynoic acid2-(2¹-methylprop-1¹-enyl)-4-pentynoic acid2-(2¹-methylprop-2¹-enyl)-4-pentynoic acid 2-tert.-butyl-4-pentynoicacid 2-n-pentyl-4-pentynoic acid 2-(1¹-methylbutyl)-4-pentynoic acid2-(2¹-methylbutyl)-4-pentynoic acid 2-(3¹-methylbutyl)-4-pentynoic acid2-(3¹-dimethylpropyl)-4-pentynoic acid2-(1¹,1¹-dimethylpropyl)-4-pentynoic acid2-(2¹,2¹-dimethylpropyl)-4-pentynoic acid 2-n-hexyl-4-pentynoic acid2-n-hex-1¹-enyl-4-pentynoic acid 2-n-hex-2¹-enyl-4-pentynoic acid2-n-hex-3¹-enyl-4-pentynoic acid 2-n-hex-4¹-enyl-4-pentynoic acid2-n-hex-5¹-enyl-4-pentynoic acid 2-(1¹-methylpentyl)-4-pentynoic acid2-(1¹-methylpent-1¹-enyl)-4-pentynoic acid2-(1¹-methylpent-2¹-enyl)-4-pentynoic acid2-(1¹-methylpent-3¹-enyl)-4-pentynoic acid2-(1¹-methylpent-4¹-enyl)-4-pentynoic acid2-(2¹-methylpentyl)-4-pentynoic acid2-(2¹-methylpent-1¹-enyl)-4-pentynoic acid2-(2¹-methylpent-2¹-enyl)-4-pentynoic acid2-(2¹-methylpent-3¹-enyl)-4-pentynoic acid2-(2¹-methylpent-4¹-enyl)-4-pentynoic acid2-(3¹-methylpentyl)-4-pentynoic acid2-(3¹-methylpent-1¹-enyl)-4-pentynoic acid2-(3¹-methylpent-2¹-enyl)-4-pentynoic acid2-(3¹-methylpent-3¹-enyl)-4-pentynoic acid2-(3¹-methylpent-4¹-enyl)-4-pentynoic acid2-(4¹-methylpentyl)-4-pentynoic acid2-(4¹-methylpent-1¹-enyl)-4-pentynoic acid2-(4¹-methylpent-2¹-enyl)-4-pentynoic acid2-(4¹-methylpent-3¹-enyl)-4-pentynoic acid2-(4¹-methylpent-4¹-enyl)-4-pentynoic acid2-(1¹,1¹-dimethylbutyl)-4-pentynoic acid2-(1¹,1¹-dimethylbut-2¹-enyl)-4-pentynoic acid2-(1¹,1¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-(1¹,2¹-dimethylbutyl)-4-pentynoic acid2-(1¹,2¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(1¹,2¹,dimethylbut-3¹-enyl)-4-pentynoic acid2-(1¹,3¹,dimethylbutyl)-4-pentynoic acid2-(1¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(1¹,2¹-dimethylbut-2¹-enyl)-4-pentynoic acid2-(1¹,3¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-(2¹,2¹-dimethylbutyl)-4-pentynoic acid2-(2¹,2¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-(2¹,3¹-dimethylbutyl)-4-pentynoic acid2-(2¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(2¹,3¹-dimethylbut-2¹-enyl)-4-pentynoic acid2-(2¹,3¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-(3¹,3¹-dimethylbutyl)-4-pentynoic acid2-(3¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(1¹,1¹,2¹-trimethylpropyl)-4-pentynoic acid2-(1¹,1¹,2¹-trimethylprop-2¹-enyl)-4-pentynoic acid2-(1¹,2¹,2¹-trimethylpropyl)-4-pentynoic acid 2-n-heptyl-4-pentynoicacid 2-)1¹-methylhexyl)-4-pentynoic acid 2-(2¹-methylhexyl)-4-pentynoicacid 2-(3¹-methylhexyl)-4-pentynoic acid 2-(4¹-methylhexyl)-4-pentynoicacid 2-(5¹-methylhexyl)-4-pentynoic acid2-(1¹,1¹-dimethylpentyl)-4-pentynoic acid2-(1¹,2¹-dimethylpentyl)-4-pentynoic acid2-(1¹,3¹-dimethylpentyl)-4-pentynoic acid2-(1¹,4¹-dimethylpentyl)-4-pentynoic acid2-(2¹,2¹-dimethylpentyl)-4-pentynoic acid2-(2¹,3¹-dimethylpentyl)-4-pentynoic acid2-(2¹,4¹-dimethylpentyl)-4-pentynoic acid2-(3¹,3¹-dimethylpentyl)-4-pentynoic acid2-(3¹,4¹-dimethylpentyl)-4-pentynoic acid2-(4¹,4¹-dimethylpentyl)-4-pentynoic acid2-(1¹,1¹,2¹-trimethylbutyl)-4-pentynoic acid2-(1¹,1¹,3¹-trimethylbutyl)-4-pentynoic acid2-(1¹,2¹,3¹-trimethylbutyl)-4-pentynoic acid2-(2¹,2¹,3¹-trimethylbutyl)-4-pentynoic acid2-(2¹,3¹,3¹-trimethylbutyl)-4-pentynoic acid.
 8. The compounds accordingto claim 7 selected from the group consisting of 2-n-butyl-4-pentynoicacid, 2-n-pentyl-4- pentynoic acid and 2-n-hexyl-4-pentynoic acid. 9.The compounds according to claim 8 wherein the compounds are selectedfrom the group consisting of 2-n-pentyl-4-pentynoic acid and2-n-hexyl-4-pentynoic acid.
 10. A method of preparing compounds havingthe general formula (I)

wherein R¹ is —C≡CH, —CH═CH₂, or —CH₂—CH₃, R² is saturated, unsaturated,branched and/or unbranched C₁₋₃₀ alkyl group, optionally substitutedwith an aliphatic of aromatic C₃₋₉ cyclohydrocarbon or heterocyclicgroup; and M is a hydrogen or a metal atom, comprising; combining amalonic acid diester with a first halide having the general formula(VII) R²—X   (VII)  wherein R² has the meaning given previously; orhalogen, to form a 2-alkyl malonic acid diester, and combining the2-alkyl malonic acid diester with a second halide having the generalformula (VIII) R¹—CH₂—X   (VIII)  wherein R¹ has the meaning givenpreviously; or  combining the malonic acid diester with the secondhalide having the general formula (VIII) to form a 2-alkyl malonic aciddiester; and  combining the first halide having the general formula(VII) to form a compound of formula (IX)

 wherein R⁵ is an alkyl group; and hydrolyzing and decarboxylating thecompound of formula (IX) to form the compound of formula (I) to a salt.11. The method according to claim 10 wherein R¹ is —C≡CH and R² is—(CH₂)₁₋₉—CH₃.
 12. The method according to claim 11 wherein R² is—(CH₂)₃₋₆—CH₃).
 13. The method according to claim 12 wherein R² is—(CH₂)₄₋₅—CH₃.
 14. The method according to claim 10 wherein the methodis used to prepare a compound selected from the group consisting of:2-n-prop-1¹-enyl-4-pentynoic acid 2-n-prop-2¹-enyl-4-pentynoic acid2-i-propyl-4-pentynoic acid 2-i-propenyl-4-pentynoic acid2-n-butyl-4-pentynoic acid 2-n-but-1¹-enyl-4-pentynoic acid2-n-but-2¹-enyl-4-pentynoic acid 2-n-but-3¹-enyl-4-pentynoic acid2-(1¹-methylbutyl)-4-pentynoic acid2-(1¹-methylprop-2¹-enyl)-4-pentynoic acid2-(1¹-methylprop-2¹-enyl)-4-pentynoic acid2-(2¹-methylpropyl)-4-pentynoic acid2-(2¹-methylprop-1¹-enyl)-4-pentynoic acid2-(2¹-methylprop-2¹-enyl)-4-pentynoic acid 2-tert.-butyl-4-pentynoicacid 2-n-pentyl-4-pentynoic acid 2-(1¹-methylbutyl)-4-pentynoic acid2-(2¹-methylbutyl)-4-pentynoic acid 2-(3¹-methylbutyl)-4-pentynoic acid2-(1¹,1¹-dimethylpropyl)-4-pentynoic acid2-(1¹,2¹-dimethylpropyl)-4-pentynoic acid2-(2¹,2¹-dimethylpropyl)-4-pentynoic acid 2-n-hexyl-4-pentynoic acid2-n-hex-1¹-enyl-4-pentynoic acid 2-n-hex-2¹-enyl-4-pentynoic acid2-n-hex-3¹-enyl-4-pentynoic acid 2-n-hex-4¹-enyl-4-pentynoic acid2-n-hex-5¹-enyl-4-pentynoic acid 2-(1¹-methylpentyl)-4-pentynoic acid2-(1¹-methylpent-1¹-enyl)-4-pentynoic acid2-(1¹-methylpent-2¹-enyl)-4-pentynoic acid2-(1¹-methylpent-3¹-enyl)-4-pentynoic acid2-(1¹-methylpent-4¹-enyl)-4-pentynoic acid2-(2¹-methylpentyl)-4-pentynoic acid2-(2¹-methylpent-1¹-enyl)-4-pentynoic acid2-(2¹-methylpent-2¹-enyl)-4-pentynoic acid2-(2¹-methylpent-3¹-enyl)-4-pentynoic acid2-(2¹-methylpent-4¹-enyl)-4-pentynoic acid2-(3¹-methylpentyl)-4-pentynoic acid2-(3¹-methylpent-1¹-enyl)-4-pentynoic acid2-(3¹-methylpent-2¹-enyl)-4-pentynoic acid2-(3¹-methylpent-3¹-enyl)-4-pentynoic acid2-(3¹-methylpent-4¹-enyl)-4-pentynoic acid2-(4¹-methylpentyl)-4-pentynoic acid2-(4¹-methylpent-1¹-enyl)-4-pentynoic acid2-(4¹-methylpent-2₁-enyl)-4-pentynoic acid2-(4¹-methylpent-3¹-enyl)-4-pentynoic acid2-(4¹-methylpent-4¹-enyl)-4-pentynoic acid2-(1¹,1¹-dimethylbutyl)-4-pentynoic acid2-(1¹,1¹-dimethylbut-2¹-enyl)-4-pentynoic acid2-(1¹,1¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-(1¹,2¹-dimethylbutyl)-4-pentynoic acid2-(1¹,2¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(1¹,2¹,dimethylbut-2¹-enyl)-4pentynoic acid2-(1¹,2¹,dimethylbut-3¹-enyl)-4-pentynoic acid2-(1¹,3¹-dimethylbutyl)-4-pentynoic acid2-(1¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(1¹,3¹-dimethylbut-2¹-enyl)-4-pentynoic acid2-(1¹,3¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-(2¹,2¹-dimethylbutyl)-4-pentynoic acid2-(2¹,2¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-(2¹,3¹-dimethylbutyl)-4-pentynoic acid2-(2¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(2¹,3¹-dimethylbut-2¹-enyl)-4-pentynoic acid2-(2¹,3¹-dimethylbut-3¹-enyl)-4-pentynoic acid2-)3¹,3¹-dimethylbutyl)-4-pentynoic acid2-(3¹,3¹-dimethylbut-1¹-enyl)-4-pentynoic acid2-(1¹,1¹,2¹-trimethylpropyl)-4-pentynoic acid2-(1¹,1¹,2¹-trimethylprop-2¹-enyl)-4-pentynoic acid2-(1¹,2¹,2¹-trimethylpropyl)-4-pentynoic acid 2-n-heptyl-4-pentynoicacid 2-)1¹-methylhexyl)-4-pentynoic acid 2-(2¹-methylhexyl)-4-pentynoicacid 2-(3¹-methylhexyl)-4-pentynoic acid 2-(4¹-methylhexyl)-4-pentynoicacid 2-(5¹-methylhexyl)-4-pentynoic acid2-(1¹,1¹-dimethylpentyl)-4-pentynoic acid2-(1¹,2¹-dimethylpentyl)-4-pentynoic acid2-(1¹,3¹-dimethylpentyl)-4-pentynoic acid2-(1¹,4¹-dimethylpentyl)-4-pentynoic acid2-(2¹,2¹-dimethylpentyl)-4-pentynoic acid2-(2¹,3¹-dimethylpentyl)-4-pentynoic acid2-(2¹,4¹-dimethylpentyl)-4-pentynoic acid2-(3¹,3¹-dimethylpentyl)-4-pentynoic acid2-(3¹,4¹-dimethylpentyl)-4-pentynoic acid2-(4¹,4¹-dimethylpentyl)-4-pentynoic acid2-(1¹,1¹,2¹-trimethylbutyl)-4-pentynoic acid2-(1¹,1¹,3¹-trimethylbutyl)-4-pentynoic acid2-(1¹,2¹,3¹-trimethylbutyl)-4-pentynoic acid2-(2¹,2¹,3¹-trimethylbutyl)-4-pentynoic acid2-(2¹¹,3¹,3¹-trimethylbutyl)-4-pentynoic acid.
 15. The method accordingto claim 10 wherein the method is used to prepare at least one compoundselected from the group consisting of 2-n-butyl-4-pentynoic acid,2-n-pentyl-4-pentynoic acid and 2-n-hexyl-4-pentynoic acid.
 16. AS-enantiomer of a compound of formula (I)

wherein R ¹ is —C≡CH, R ² is a saturated, unsaturated, branched and/orunbranched C ₁ -C ₃₀ alkyl group, optionally further comprising analiphatic or aromatic C ₃₋₉ cyclohydrocarbon or heterocyclic groupcomprising 3 to 9 atoms; and M is a hydrogen or a metal atom, andpharmaceutical acceptable salts thereof; with the proviso that thecompound of formula (I) is not 2 -n-propyl- 4 -pentynoic acid.
 17. Acompound according to claim 16 wherein R² is —(CH ₂)₃₋₆ —CH ₃ .
 18. Acompound according to claim 16 wherein R² is —(CH ₂)₄₋₅ —CH ₃ .
 19. Acompound according to claim 16 selected from the group consisting of: 2-n-prop- 1 ¹-enyl- 4 -pentynoic acid 2 -n-prop- 2 ¹-enyl- 4 -pentynoicacid 2 -i-propyl- 4 -pentynoic acid 2 -i-propenyl- 4 -pentynoic acid 2-n-butyl- 4 -pentynoic acid 2 -n-but- 1 ¹-enyl- 4 -pentynoic acid 2-n-but- 2 ¹-enyl- 4 -pentynoic acid 2 -n-but- 3 ¹-enyl- 4 -pentynoicacid 2 -( 1 ¹-methylbutyl)- 4 -pentynoic acid 2 -( 1 ¹-methylprop- 1¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹-methylprop- 2 ¹-enyl)- 4 -pentynoicacid 2 -( 2 ¹-methylpropyl)- 4 -pentynoic acid 2 -( 2 ¹-methylprop- 1¹-enyl)- 4 -pentynoic acid 2 -( 2 ¹-methylprop- 2 ¹-enyl)- 4 -pentynoicacid 2 -tert.-butyl- 4 -pentynoic acid 2 -n-pentyl- 4 -pentynoic acid 2-( 1 ¹-methylbutyl)- 4 -pentynoic acid 2 -( 2 ¹-methylbutyl)- 4-pentynoic acid 2 -( 3 ¹-methylbutyl)- 4 -pentynoic acid 2 -( 1 ¹ ,1¹-dimethylpropyl)- 4 -pentynoic acid 2 -( 1 ¹ ,2 ¹-dimethylpropyl)- 4-pentynoic acid 2 -( 2 ¹ ,2 ¹-dimethylpropyl)- 4 -pentynoic acid 2-n-hexyl- 4 -pentynoic acid 2 -n-hex- 1 ¹-enyl- 4 -pentynoic acid 2-n-hex- 2 ¹-enyl- 4 -pentynoic acid 2 -n-hex- 3 ¹-enyl- 4 -pentynoicacid 2 -n-hex- 4 ¹-enyl- 4 -pentynoic acid 2 -n-hex- 5 ¹-enyl- 4-pentynoic acid 2 -( 1 ¹-methylpentyl)- 4 -pentynoic acid 2 -( 1¹-methylpent- 1 ¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹-methylpent- 2¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹-methylpent- 3 ¹-enyl)- 4 -pentynoicacid 2 -( 1 ¹-methylpent- 4 ¹-enyl)- 4 -pentynoic acid 2 -( 2¹-methylpentyl)- 4 -pentynoic acid 2 -( 2 ¹-methylpent- 1 ¹-enyl)- 4-pentynoic acid 2 -( 2 ¹-methylpent- 2 ¹-enyl)- 4 -pentynoic acid 2 -( 2¹-methylpent- 3 ¹-enyl)- 4 -pentynoic acid 2 -( 2 ¹-methylpent- 4¹-enyl)- 4 -pentynoic acid 2 -( 3 ¹-methylpentyl)- 4 -pentynoic acid 2-( 3 ¹-methylpent- 1 ¹-enyl)- 4 -pentynoic acid 2 -( 3 ¹-methylpent- 2¹-enyl)- 4 -pentynoic acid 2 -( 3 ¹-methylpent- 3 ¹-enyl)- 4 -pentynoicacid 2 -( 3 ¹-methylpent- 4 ¹-enyl)- 4 -pentynoic acid 2 -( 4¹-methylpentyl)- 4 -pentynoic acid 2 -( 4 ¹-methylpent- 1 ¹-enyl)- 4-pentynoic acid 2 -( 4 ¹-methylpent- 2 ¹-enyl)- 4 -pentynoic acid 2 -( 4¹-methylpent- 3 ¹-enyl)- 4 -pentynoic acid 2 -( 4 ¹-methylpent- 4¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹- 1 ¹-dimethylbutyl)- 4 -pentynoicacid 2 -( 1 ¹- 1 ¹-dimethylbut- 1 ¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹- 1¹-dimethylbut- 3 ¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹- 2¹-dimethylbutyl)- 4 -pentynoic acid 2 -( 1 ¹- 2 ¹-dimethylbut- 1¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹- 2 ¹-dimethylbut- 2 ¹-enyl)- 4-pentynoic acid 2 -( 1 ¹- 2 ¹-dimethylbut- 3 ¹-enyl)- 4 -pentynoic acid2 -( 1 ¹- 3 ¹-dimethylbutyl)- 4 -pentynoic acid 2 -( 1 ¹- 3¹-dimethylbut- 1 ¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹- 3 ¹-dimethylbut- 2¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹- 3 ¹-dimethylbut- 3 ¹-enyl)- 4-pentynoic acid 2 -( 2 ¹- 2 ¹-dimethylbutyl)- 4 -pentynoic acid 2 -( 2¹- 2 ¹-dimethylbut- 3 ¹-enyl)- 4 -pentynoic acid 2 -( 2 ¹- 3¹-dimethylbutyl)- 4 -pentynoic acid 2 -( 2 ¹- 3 ¹-dimethylbut- 1¹-enyl)- 4 -pentynoic acid 2 -( 2 ¹- 3 ¹-dimethylbut- 2 ¹-enyl)- 4-pentynoic acid 2 -( 2 ¹- 3 ¹-dimethylbut- 3 ¹-enyl)- 4 -pentynoic acid2 -( 3 ¹- 3 ¹-dimethylbutyl)- 4 -pentynoic acid 2 -( 3 ¹- 3¹-dimethylbut- 1 ¹-enyl)- 4 -pentynoic acid 2 -( 1 ¹- 1 ¹- 2¹-trimethylpropyl)- 4 -pentynoic acid 2 -( 1 ¹- 1 ¹- 2 ¹-trimethylprop-2 -enyl)- 4 -pentynoic acid 2 -( 1 ¹- 2 ¹- 2 ¹-trimethylpropyl)- 4-pentynoic acid 2 -n-heptyl- 4 -pentynoic acid 2 -( 1 ¹-methylhexyl)- 4-pentynoic acid 2 -( 2 ¹-methylhexyl)- 4 -pentynoic acid 2 -( 3¹-methylhexyl)- 4 -pentynoic acid 2 -( 4 ¹-methylhexyl)- 4 -pentynoicacid 2 -( 5 ¹-methylhexyl)- 4 -pentynoic acid 2 -( 1 ¹ ,1¹-dimethylpentyl)- 4 -pentynoic acid 2 -( 1 ¹ ,2 ¹-dimethylpentyl)- 4-pentynoic acid 2 -( 1 ¹ ,3 ¹-dimethylpentyl)- 4 -pentynoic acid 2 -( 1¹ ,4 ¹-dimethylpentyl)- 4 -pentynoic acid 2 -( 2 ¹ ,2 ¹-dimethylpentyl)-4 -pentynoic acid 2 -( 2 ¹ ,3 ¹-dimethylpentyl)- 4 -pentynoic acid 2 -(2 ¹ ,4 ¹-dimethylpentyl)- 4 -pentynoic acid 2 -( 3 ¹ ,3¹-dimethylpentyl)- 4 -pentynoic acid 2 -( 3 ¹ ,4 ¹-dimethylpentyl)- 4-pentynoic acid 2 -( 4 ¹ ,4 ¹-dimethylpentyl)- 4 -pentynoic acid 2 -( 1¹ ,1 ¹ ,2 ¹-trimethylbutyl)- 4 -pentynoic acid 2 -( 1 ¹ ,1 ¹ ,3¹-trimethylbutyl)- 4 -pentynoic acid 2 -( 1 ¹ ,2 ¹ ,3 ¹-trimethylbutyl)-4 -pentynoic acid 2 -( 2 ¹ ,2 ¹ ,3 ¹-trimethylbutyl)- 4 -pentynoic acid2 -( 2 ¹ ,3 ¹ ,3 ¹-trimethylbutyl)- 4 -pentynoic acid.
 20. A compoundaccording to claim 19 wherein the compounds are selected from the groupconsisting of 2-n-pentyl- 4 -pentynoic acid and 2 -n-hexyl- 4 -pentynoicacid.
 21. 2-n-pentyl- 4 -pentynoic acid and enantiomers thereof andpharmaceutically acceptable salts thereof.
 22. A S-enantiomer of thecompounds of claim 21 and pharmaceutically acceptable salts thereof. 23.A method of preparing a compound having the general formula (I)

wherein R ¹ is —C≡CH, R ² is a saturated, unbranched C ₅ alkyl group;and M is a hydrogen or a metal atom, comprising: combining a malonicacid diester with a first halide having the general formula (VII) R ²—X  (VII)  wherein R ² has the meaning given previously to form a 2-pentyl malonic acid diester, and combining the 2 -pentyl malonic aciddiester with a second halide having the general formula (VIII) R ¹ —CH ₂—X   (VIII)  wherein R ¹ has the meaning given previously; or combiningthe malonic acid diester with the second halide having the generalformula (VIII) to form a 2 -pentyl malonic acid diester; and combiningthe first halide having the general formula (VII) to form a compound offormula (IX)

 wherein R ⁵ is an alkyl group; and hydrolyzing and decarboxylating thecompound of formula (IX) to form the compound of formula (I); andoptionally converting the compound of formula (I) to a salt.